Method and apparatus for the centrifugal washing of particles in a closed system



1967 D. BELLAMY. JR. ETAL 3,347,454

METHOD AND APPARATUS FOR THE CENTRIFUGAL WASHING OF PARTICLES IN A CLOSED SYSTEM Filed May 13, 1964 2 Sheets-Sheet 1 1 F F m N J 5 c 8 a: m

O U q- Q 2 o 2 J J l .I 1?; w 55 0 N Ll- 52 Q g g (D N INVENTOR. 5 3 DAVID BELLAMY JRHA]. BY

ATTORNEY Oct. 17, 1967 .BELLAMY. JR. ETAL 3,347,454

METHOD AND APPARATUS FOR THE CENTRIFUGAL WASHING OF PARTICLES IN A CLOSED SYSTEM Filed May 13, 1964 2 Sheets-Sheet 2 200 E l5u INVENTOR. DAVID BELLAMY JRHAL ATTORNEY United States Patent Ofitice Glenview, IIHL, Morton The present invention relates to a novel apparatus and method for washing particles. More particularly, it relates to an apparatus and method for the washing of particles within a substantially closed and disposable system in which the particles are maintained in the washing field by centrifugal force.

Recently, there has developed the need, in various fields relating to the biological sciences, for an apparatus and a method of washing tiny particles, such as biological cells, within a substantially closed system, to minimize the risk of contamination. One area of activity in which such a need is particularly urgent is the area which relates to the preservation of blood.

In recent years, various of the researchers in the aforementioned area of activity have proposed new techniques for extending the storage life of human blood from the present twentyone days maximum to an indefinite period of time. The proposed techniques generally comprise adding a preservative to the blood and then freezing the blood until time of use. The success of the newly proposed techniques is generally believed to be dependent upon two factors: the first, finding a preservative, which can become sufficiently concentrated within the blood cells to protect the cells from physical damage during the periods of freezing, storage and thawing; and, the second, effectively removing the preservative prior to the transfusion of the blood.

The first problem, that of finding a suitable preservative agent, has been solved to a large extent by the discovery that glycerol, polyvinyl pyrrolidone and/or dimethyl sulfoxide become sufliciently concentrated within blood cells to prevent the aforementioned physical damage to the cells. On the other hand, the second problem, that of removing the preservative has up to the present time only been accomplished with difficulty and by time consuming techniques.

The availability of an apparatus and method for quickly and adequately washing impurities from red blood cells and similar particles Within a closed system could and would substantially contribute to the further evaluation and success of the above mentioned and additional techniques.

It is, therefore, an object of the present invention to disclose a novel apparatus and method for washing particles, such as red blood cells, within a substantially closed system.

It is a further object of the present invention to disclose a novel method and apparatus for simultaneously washing and centrifuging such particles.

It is a still further object of the present invention to disclose a novel disposable container for washing such particles.

It is an additional object of the present invention to disclose a novel disposable wash fluid distribution means, which makes possible the continuous washing of the particles during centrifugation.

These and still further objects will become apparent from the following description of a preferred embodiment and the accompanying drawings, wherein:

FIGURE 1 is a vertical section showing one embodiment of the novel apparatus of the present invention.

3,347,454 Patented Oct. 17, 1.967

FIGURE 2 is an enlarged section view of the novel fluid distribution means of the present invention.

FIGURE 3 is an elevati-onal view of one embodiment of the novel disposable container of the: present invention.

FIGURE 4- is an enlarged view of a retaining means employed in the preferred embodiment of the present invention.

In FIGURE 1, there is illustrated a vertical section of a rotary type centrifuge 1. The spindle 10 of the centrifuge supports of rotor 11, which is provided with container receiving cups or buckets 12. Positioned within the buckets 12 are the disposable, flexible, collapsible, con tainers 13, which are provided with an integral inlet tube 14 and an integral outlet tube 15. The containers 13 are maintained in the desired position within the buckets 12 by retaining means 16.

Communication with the interior of the containers 13 is had through the integral inlet tube 14 and the integral outlet tube 15, which lead from the plastic containers and through the retaining means 16 to the rotary seal 17. To provide communication with the interior of the seal, the free ends of inlet tube 14 and the outlet tube 15 are joined to the inlet connection 14a and the outlet connection 15a of the rotary seal 17.

The rotary seal 17, as illustrated in FIGURE 2, is comprised of a hollow cylindrical housing 18 and a stationary shaft 19. A major portion of the stationary shaft 19 is positioned within the inner chamber of the housing 18 of the rotary seal and maintained in such position by a pair of seals 20 and 21. The seals 20 and 21 are spaced apart and cooperate with the shaft to divide the interior of the housing 18 into two distinct compartments, 20a and 2101, respectively. The seals 20 and 21 frictionally engage the stationary shaft 19 and maintain the compartments, 20a and 21a, fluid tight and distinct from one another.

As seen in FIGURE 2, the stationary shaft 19 is provided with the passageways 22 and 23. The passageway 22 extends through the stationary shaft 19 and communicates only with compartment 21a. Passageway 23, which comprises the remainder of the interior of the hollow stationary shaft 19 opens into and communicates only with compartment 20a.

The housing 18, is further provided with means 24 for securing it to the rotor, in the manner seen in FIGURE 1. When thus secured, the housing 18 rotates freely with the rotor. The shaft 19 is maintained in a stationary position by stabilizing means 25, thereby preventing it from rotating with the rotor. The means 25 may take various forms, for example, the bracket seen in FIGURE 1.

In the preferred embodiment of the invention, the centrifuge employed is a rotary type refrigerated centrifuge, such as that sold under the trademark Servall RC-3 by Ivan So-rvall, Inc. of Norwalk, Conn. The rotor of the preferred centrifuge has four container receiving compartments which are provided with 600 ml. stainless steel cups with a plastic coated interior. The angle of inclination of these cups is 34 from the vertical. This centrifuge is capable of 5,000 revolutions per minute and is provided with adequate temperature controls to prevent damage to the red blood cells as the result of over-heating.

The preferred centrifuge is provided with means such as a threaded female member in the center of the rotor which enables the rotary seal to be attached. The centrifuge is also provided with openings for the tubes 26 and 27 which allow for the introduction and removal of a sterile wash fluid. In addition to the described rotary type centrifuge, a variety of other centrifuges may be used, such as the swinging bucket type centrifuge.

In the preferred embodiment, the rotary seal 17 is formed of suitably inexpensive materials to allow it to be discarded after a single use. Preferably, the housing 18 is a generally cylindrical plastic member with an interior chamber having a diameter of inch, and a depth of 1 /2 inches. Communicating with the interior chamber are the inlet and outlet connections 14a and a, respectively. When employed with the previously described centrifuge, the rotary seal is equipped with four separate inlet connections 14a and an equal number of outlet connections 15a to provide for the introduction of wash fluid into and the removal of wash fluid from a container positioned in each of the container-receiving cups.

Preferably, the stationary shaft 19 has an outer diameter of approximately /s inch. Located at the bottom of the shaft is the exit of the passageway 22 which extends throughout the length of the shaft and communicates with the wash fluid inlet tube 26. Intermediate the length of the shaft is the entrance of the passageway 23. Passageway 23 comprises the remainder of the interior of the otherwise hollow stationary shaft and completely surrounds the passageway 22. The shaft 19 may be formed completely of a single material such as steel, but, preferably a combination of materials, such as rubber, metal and plastic is used to provide the optimum resistance to wear and leakage at the lowest possible cost.

While in the preferred embodiment the shaft 19 has been described as stationary and the housing 18 as rotary, it will be readily understood by those skilled in the art that the housing can be made stationary and the shaft can be made rotary, if the two parts are inverted and reversed.

The seals and 21 are formed of a resilient material, such as plastic or rubber. Preferably they are shaped to conform to and cooperate with the outer surface of the stationary shaft so as to prevent the flow of fluid between the shaft and the seal. These seals are fastened within the housing 18 by any suitable means, such as press fitting, welding, heat sealing, gluing or the like. In the preferred embodiment, the seals are circular in shape having an outer diameter of inch and an inner diameter of /8 inch, and, they are positioned /2 and 1% inches from the interior bottom of the housing chamber, respectively.

The flexible, plastic containers 13 are preferably formed of plastic material. In the preferred form they are formed of polyvinylchloride resin, which is hemorepellent; and they are sterilized prior to use. As seen in FIGURE 3, they take the shape of a uniform oval having a maximum length of six inches and a minimum width of 3%. inches. Heat sealed into communication with the interior of the container are the inlet and outlet tubes 14 and 15, respectively. The portion of the tube 14 which extends within the interior of the container 13 is provided with sufficient perforations 28 to promote and stimulate the agitation of the particles to be Washed within the container by the inflow of wash fluid ino the container. The overall length of the tube 15 and that portion of the tube 14 which extends outside the container is not critical and need only be such that it will provide for direct or indirect connection to the inlet and outlet connections of the housing 18 Without interfering with the operation of the apparatus.

When it is intended to employ the containers 13 for the washing of red blood cells, the container may be modified to allow it to serve as an original blood donation receiving container. Such modifications might simply comprise equipping the inlet tube 14- with a donor needle, and the outlet tube 15 with a suitable protective closure (e.g. such as that disclosed in US. Patent No. 2,894,510). Upon the collection of a blood donation the inlet tube 14 may be heat sealed to provide a hermetically sealed container. The tube 14 could later, at time of use, be reopened for attachment to the inlet tube connection 14a.

In the described embodiment, the retaining means, the container-receiving cup and the bag all cooperate to shape and locate the bag so as to provide for the optimum outletting of the fluid. This desired result is generally achieved when the outlet tube is positioned closer to the axis of rotation of the centrifuge than any other portion of the bag. When the outlet tube is so positioned the likelihood of the existence of dead spots, which contain incompletely or unwashed particles, is substantially reduced.

In other embodiments of the invention, as for example, Where the position or shape of the cups, or the shape of the bags is materially varied, it will generally be necessary to employ differently shaped retaining means to obtain the desired optimum outletting of the fluid.

When the centrifuge and the plastic containers which have been specifically described are employed, the retaining means 16 (as best seen in FIGURE 4) takes the form of an oval having a maximum length of 5 /8 inches and a maximum width of 3 /8 inches. The notches 16a and 16b are cut or otherwise formed of an adequate size to provide for the outer diameter of tube 14 and 15. Preferably, the retaining means 16 is a metal plate of sufficient weight and suitable shape to prevent the pressure, which is built up within the plastic containers by the fluid distribution system, from rupturing the inner-most wall of the container.

From the foregoing discussion it is understandable that the exact dimensions, design and number of the various previously described components will be influenced by the number and the shape of the container receiving cups of the centrifuge, as well as still other factors.

When they are employed for the removal of impurities, such as preservatives from blood, the plastic containers and their contents are placed within the container-receiving cups or buckets with the inlet and outlet tubes extending toward the axis of the centrifuge. The retaining means 16 is then placed into position with the inlet tube 14 and the outlet tube 15 extending through the notches Ma and 16b, respectively. The free ends of the inlet and outlet tubes 14 and 15 are then attached to the inlet and outlet connections 14a and 15a of the rotary seal, respectively. The rotary seal is then secured to the rotor and the stationary shaft 1? of the rotary seal is stabilized by the means of the bracket or other stabilizing means 25. Centrifugation is then commenced and a sterile washing fluid is introduced under gravity pressure or pumping via the Wash fluid inlet tube 26. The fluid passes through the wash fluid inlet tube 26 and the passageway 22 of the shaft into the lower compartment of the rotary seal 21a. It then leaves the shaft and flows via the inlet tube 14 into the plastic container 13. After the wash fluid enters the interior of the container 13, it leaves the inlet tube through the perforations 28 in the form of a myriad of tiny streams which agitate the particles in the container. Agitation of the particles by the incoming Wash fluid cooperates with the centrifugal force to maintain the particles in a more or less constant state of agitation in an area adjacent the bottom of the bucket or cup, thereby allowing for the removal of the spent wash fluid substantially free of cells via the outlet tube 15. 'Upon leaving the container, the spent wash fluid travels via the outlet tube 15 to the upper compartment Zita of the rotary seal. It then enters the interior of the stationary shaft 19, via the passageway 23, and flows about the exterior of the passageway 22, and out of the shaft through wash fluid discard tube 27. The circulating of the spent wash fluid through the stationary shaft 19 cools the shaft, and, thereby effectively prevents the seals 20 and 21 from being destroyed by the heat that generates from the frictional contact with the shaft.

In the foregoing discussion, the novel apparatus and method of the present invention have been described in detail in connection with the washing of red blood cells free of preservatives. It is to be understood, however, that the invention can be employed with considerable success for washing a wide variety of particles, including other types of biological cells, human formed elements, and the like, free of an equally wide variety of impurities, such as bacteria, viruses and the like.

From the foregoing description, it will also be readily apparent to those skilled in the art that the present invention provides many advantages over the prior art devices.

For example, it permits the simultaneous washing of the contents of a plurality of containers of equal volume without a co-mingling or cross contamination; and, it allows the washing to be done in a disposable and substantially closed system.

Still other advantages not described herein will be apparent to those skilled in the art.

The foregoing description of a preferred embodiment has been merely for purposes of illustration and it should be understood that a wide variety of changes and modifications may be made without departing from the spirit and scope of the present invention.

What is claimed is:

1. The method of washing particles within a substantially closed system which comprises, placing the particles to be washed into a disposable flexible, collapsible, container, placing said container within the container-receiving cup of a centrifuge, and then centrifuging said container and its contents while introducing a wash fluid at the bottom of said container to wash said particles.

2. The method of simultaneously washing and centrifuging particles within a substantially closed system which comprises placing the particles within a disposable flexible, collapsible, container, placing the container within the container-receiving cup of a centrifuge, centrifuging said container and its contents while introducing a wash fluid at the bottom of said container to wash said particles and then removing the spent wash fluid.

3. An apparatus for washing particles within a substantially closed and disposable system, which comprises a centrifuge having container-receiving cups, at least one flexible, collapsible container adapted to contain unwashed particles, a container positioned in at least one of said cups, and means for introducing a wash fluid at the bottom of said container and for removing spent wash fluid from said container during centrifugation.

4. An apparatus for washing particles within a substantially closed and disposable system, which comprises, a centrifuge provided with container-receiving cups, a flexible, collapsible container adapted to contain unwashed particles positioned within one of said cups, said container being provided with an integral inlet tube extending to the bottom of the container and an integral outlet tube, retaining means positioned in said cup and retaining and shaping said container to provide for the optimum outletting of fluid from said container via said outlet tube and means for introducing a wash fluid into and removing it from said container during centrifugation.

5. The apparatus of claim 4 wherein the retaining means is a weight means on said collapsible container, said weight means resting on said collapsible container and slidable within said cup.

6. An apparatus for washing particles within a substantially closed and disposable system which comprises, a centrifuge provided with a plurality of container-receiving cups, a plurality of flexible, collapsible containers adapted to contain unwashed particles positioned within said cups said containers being provided with an integral inlet tube extending to the bottom of the container and an integraI outlet tube, and means for introducing and removing 2 wash fluid from said containers via said inlet tube and outlet tube during centrifugation, said means comprising 2 rotary seal which provides for the introduction of sterile wash fluid and the removal of spent wash fluid from each of said plurality of containers without a co-mingling and a cross-contamination of the containers contents.

7. An apparatus for washing particles within a substantially closed and disposable system, which apparatus comprises, in part, a disposable, collapsible, flexible container adapted to contain the particles to be washed, said container being provided with an integral inlet tube and an integral outlet tube, said inlet tube having a portion thereof extending into the interior of said container and adapted to be positioned at the lowest point of the container when in use, said portion being provided with a plurality of perforations so that fluid entering said container will leave said tube as a myriad of small streams.

8. A rotary seal comprising a housing having an inlet and outlet connection, a shaft provided with a pair of passageways coaxially positioned within said housing and spaced therefrom, and a pair of spaced apart seals cooperating with said shaft in dividing the interior of the housing into two compartments, each of which compartments communicates with only one passageway of the shaft and a connection on said housing.

9. An apparatus for washing particles within a substantially closed and disposable system, which apparatus comprises a disposable, collapsible, flexible container adapted to contain the particles to be washed, said container being provided with an inlet tube and an outlet tube, said inlet tube having a portion thereof extending into the interior of said container and. adapted to be positioned at the most remote part of the container, relative to the centrifuge axis when in use, said portion being provided with distribution means for distributing the fluid entering said container.

References Cited UNITED STATES PATENTS 1,644,492 10/ 1927 Rawolle 233-14 2,867,582 1/ 1959 Shuman et al. 3,050,238 8/1962 Doyle et a1. 233-15 3,145,713 8/ 1964 Latham 233-22 3,211,368 10/1965 Shanley 233-26 FOREIGN PATENTS 772,030 8/1934 France. 864,410 4/ 1961 Great Britain.

M. CARY NELSON, Primary Examiner. HENRY T. KLINKSIEK, Examiner. 

1. THE METHOD OF WASHING PARTICLES WITHIN A SUBSTANTIALLY CLOSED SYSTEM WHICH COMPRISES, PLACING THE PARTICLES TO BE WASHED INTO A DISPOSABLE FLEXIBLE, COLLAPSIBLE, CONTAINER, PLACING SAID CONTAINER WITHIN THE CONTAINER-RECEIVING CUP OF A CENTRIFUGE, AND THEN CENTRIFUGING SAID CONTAINER AND ITS CONTENTS WHILE INTRODUCING A WASH FLUID AT THE BOTTOM OF SAID CONTAINER TO WASH SAID PARTICLES. 